Modification of the hepatotoxic effect of chloroform and halothane in calves

The result of the present study has clearly shown that pretreatment with phenobarbitone increases the activity of one of the drug-metabolising enzymes, i

biopsied to assess the levels of amidopyrine-N-demethylase in their livers. TWO phenobarbitone-treated and two control calves were given a single dose of 30 ml of chloroform mixed with 30 ml of liquid paraffin by stomach tube (expt I); four phenobarbitone-treated and four control calves were given 45 ml of halothane mixed with 45 ml of liquid paraffi by stomach tube (expt 11).
All calves in experiments I and I1 were biopsied 2 days after the administration of chloroform or halothane and again a week later. Liver samples were divided into three portions and were used for histological, histochemical and biochemical studies. All calves were bled daily for 14 days after the administration of the hepatotoxic drug.

Histological methods
The portion of each liver biopsy sample for histological study was fixed in 4 per cent. buffered formaldehyde in saline; 5-pm-thick paraffin sections were stained with haematoxylin and eosin (HE), by Foot's silver impregnation method and with Van Gieson's mixture for reticulin and collagen fibres, by the periodic acid-Schiff (PAS) method with and without prior incubation with diastase, and with phosphotungstic acid-haematoxylin (PTAH) for particulate cytoplasmic staining.

Histochemical methods
For this study a portion of each liver biopsy sample was immediately immersed in isopentane cooled to -65°C and stored at -70°C until sectioned at 8-pm thickness in a cryostatic microtome kept at -18°C. Glass-mounted sections were processed for the demonstration of succinic tetrazoleum reductase, glutamic dehydrogenase, non-specific esterase, adenosine triphosphatase and glucose-6-phosphatase (Pearse, 1960).

Biochemical methodr
The third portion of each liver biopsy (about 200400 mg) was weighed and homogenised for 10 s in cold 0 . 1 5~ potassium chloride with an Ultraturrax tissue grinder (Janke and Kunkel, K.G.). The supernatant collected after centrifugation at 1000 g for 30 min. was assayed for amidopyrine-N-demethylase activity by the method of McLean and McLean (1966).
Blood samples were allowed to clot and the separated sera were stored at -20°C until analysed. Concentrations of bilirubin and of glutamate-oxalacetic transaminase (GOT), arginase (ARG), sorbitol dehydrogenase (SD) and glutamate dehydrogenase (GD) were determined by methods given by Gopinath and Ford (1969).

Clinical disturbances
After the administration of both chloroform and halothane the calves became dull and narcotised for a few hours and then recovered rapidly. One of the calves from experiment 11 that had been pretreated with phenobarbitone died 2 hr after the administration of halothane.
Histological changes Experiment I. There were no histological lesions in any of the biopsy samples taken before the administration of chloroform. The liver samples taken from two calves with no pretreatment 2 days after the administration of chloroform showed only slight eosinophilia of a few of the centrilobular cells. However, the two calves that were pretreated with 14 daily doses of phenobarbitone and then given chloroform had marked centrilobular necrosis with haemorrhage ( fig. 1). Many cells in the adjacent zones were ballooned, the central sinusoids contained many polymorphonuclear neutrophils and the cells around the portal areas showed no change. There was mai-ked reduction in PAS-positive staining, especially in the centrilobular areas of necrosis and surrounding midzonal cells, and there was reduction in particulate staining in the centrilobular cells in the corresponding section stained with PTAH. The liver biopsy samples taken 9 days after chloroform dosage showed little change Experiment II. The post-dosing biopsy samples taken 2 days after the administration of halothane alone to four calves showed no structural alterations. In contrast, the four calves that received phenobarbitone and halothane had marked histological changes in the liver. The calf that died 2 hr after the administration of halothane showed focal areas of degenerated swollen liver cells and congested sinusoids. The biopsy samples taken 2 days l o L S D 0 5L-L /--"2,.. after the halothane from the remaining three calves had irregularly distributed foci of swelling, ballooning and cell necrosis ( fig. 2). Cells adjacent to these foci were vacuolated and showed increased cytoplasmic eosinophilia; some also contained refractive eosinophilic inclusions in the cytoplasm. The sinusoids in these areas had an increased neutrophil content. The PAS preparation showed a focal depletion of glycogen staining ( fig. 3) and many neighbouring cells contained PAS-positive diastase-resistant cytoplasmic inclusions.
The PTAH preparations showed a depletion of particulate staining in the focal areas of degenerating cells. There were no histological changes in any of the biopsy samples taken 9 days after halothane administration.  x 100.
Histochemistry Experiment I. The liver samples taken from the two calves 2 days after dosing with chloroform alone showed a slight reduction in canalicular ATPase activity of the centrilobular cells. There was no significant change in any other enzyme preparations. The samples taken 2 days after chloroform dosing from the two calves pretreated with phenobarbitone showed marked reduction of activity of the following enzymes in centrilobular cells ; succinic tetrazolium reductase, glutamic dehydrogenase ( fig. 4), non-specific esterase, and glucose-6-phosphatase. ATPase was absent from canaliculi of the central and midzonal liver cells ( fig. 5). There was a marked increase in diffuse cytoplasmic deposits. The liver samples taken 9 days after the administration of chloroform to two calves without phenobarbitone pretreatment had a normal histochemical distribution of enzymes. The samples from the calves pretreated with phenobarbitone still showed some reduction in the a activity of canalicular ATPase, succinic tetrazolium reductase, G D and glucose-6-phosphatase and non-specific esterase from the centrilobular liver cells.
Experiment II. The liver samples from four calves taken 2 and 9 days after treatment with halothane alone had no changes in any of the histochemical enzyme preparations. The biopsy samples taken from the three surviving phenobarbitone-pretreated calves 2 days after halothane showed focal loss of succinic tetrazolium reductase, GD, non-specific esterase ( fig. 6), and glucose-6-phosphatase activity. In groups of cells there was loss of canalicular , ? I ATPase activity with some increase in diffuse deposits. The biopsy samples that were taken 9 days after the halothane dose exhibited no changes in enzyme histochemistry.

Changes in amidopyrine-N-demethylase in calf liver
As shown in figs. 7 and 8 the enzyme content of the liver of the calves dosed with phenobarbitone in both experiments I and IT rose to a level of 30-40 pg 4-aminoantipyrine formed in 30 min. per g of liver. These raised levels fell sharply following the administration of either chloroform or halothane to the calves.
Changes in the serum constituents Experiment I. The concentration of bilirubin in serum showed no alteration in any of the calves. Administration of chloroform alone caused a slight increase in the concentration of GD in serum. There was no significant leakage of ARG, SD and GOT ( fig. 9). Adminis-tration of chloroform to the two phenobarbitone-pretreated calves resulted in marked leakage of ARC, GD, SD and GOT ( fig. 10).  . 11). In contrast, the three calves that received phenobarbitone and halothane showed considerable leakage of SD, GD and GOT into the serum ( fig. 12), although there was no significant change in the concentration of bilirubin in serum.

DISCUSSION
Single oral doses of chloroform or halothane to calves failed to produce any significant histological lesion in the liver, whereas in the horse, similar doses of chloroform produced marked centrilobular necrosis (Wolff, Lumb and Ramsay, 1967;Thorpe et al., 1969), with accompanying release of liver specific enzymes into the circulation. Similarly Gopinath et al. (1970) showed that the oral administration of halothane to horses produced centrilobular degeneration and necrosis with accompanying release of enzymes. The absence of such lesions in the calf suggests that there is a marked species difference in susceptibility to hepatotoxic agents. The lack of significant histological change due to chloroform or halothane alone was confirmed by the histochemical studies, which showed that only canalicular ATPase was slightly reduced in the centrilobular cells after chloroform.
Part of the hepatotoxic effect of chloroform has been attributed to the synthesis of toxic metabolites by Scholler (1968), Van Dyke (1969 and McLean (1970). Induction of drugmetabolising enzymes in the liver by agents such as phenobarbitone increases susceptibility to the toxic effects of chloroform in rats (McLean; Scholler, 1970). Honjo and Netter (1969) have shown that the toxicity of chloroform in the rat can be reduced by pretreatment with the enzyme-inhibitor disulfuram. Ethoxyquin given to sheep before the administration of carbon tetrachloride prevented its hepatotoxicity (Cawthorne et al., 1971).
Halothane is also partly metabolised by liver (Van Dyke, 1966), but enhancement of its toxicity had not previously been reported. The irregular distribution of the necrotic foci in the liver of the calves treated with phenobarbitone and halothane is of interest. The halothane lesion in the liver of the horse was centrilobular (Gopinath et al.) and several other liver lesions of horses, cattle and sheep all had a definite lobular localisation . The cause of this irregular distribution is not clear, but could be investigated by histochemical studies of the localisation of drug-metabolising enzymes.
The result of the present study has clearly shown that pretreatment with phenobarbitone increases the activity of one of the drug-metabolising enzymes, i.e., amidopyrine-Ndemethylase, and that the liver of calves so treated is more susceptible to the toxic effects of chloroform and of halothane as demonstrated by histological, histochemical and biochemical studies. The rare instances of hepatotoxicity following the administration of chloroform or halothane to animals may arise from previous exposure to drugs that have stimulated the drug metabolising enzymes of the liver.
The development of resistance to the repeated weekly administration of carbon tetrachloride to sheep (Ford and Lawrence, 1965) chloroform to horses (Thorpe et al., 1969) and halothane to horses (Gopinath et al.) is an interesting phenomenon. The sharp reduction in the level of amidopyrine-N-demethylase following the administration of chloroform or halothane to the calves in the present experiment suggests that the DME synthesis may progressively be reduced in these cases of repeated liver injury. This reduced DME activity could explain the reduced susceptibility to the repeated administration of carbon tetrachloride, chloroform and halothane. This possibility is now being investigated.

SUMMARY
The administration to calves of 30 ml of chloroform or 45 ml of halothane by stomach tube produces no structural alterations in the liver and either no change (halothane) or only a slight reduction in the canalicular ATPase activity of the centrilobular cells (chloroform). The same treatment given after a course of 14 daily oral doses of phenobarbitone sodium (20 mg per kg) causes centrilobular necrosis and haemorrhage (chloroform) or irregularly distributed foci of swollen and ballooned cells (halothane). With both poisons there is loss of glycogen (particularly from the swollen and ballooned cells in halothane poisoning), and of particulate staining. There is also a reduction in the activity of several enzymes in the centrilobular zone (chloroform) or in scattered foci (halothane), and release of liver-specific enzymes into the plasma, more marked with halothane.
The rise in amidopyrine-N-demethylase produced in liver by the phenobarbitone treatment was rapidly reversed by administration of chloroform or halothane.
We are grateful to the Wellcome Trust for financial support, to Miss J. Evans and Mrs J. Weston for technical assistance, and to Messrs G. Weston and A. Hesketh for photography.